Bacterial biofilms are able to form on almost any surface. Because of the impact that biofilms have on diverse environments, from indwelling devices in hospital patients to water pipes, there has been a great interest in investigating the molecular mechanisms underlying biofilm formation and maintenance. As a consequence we now know a large amount regarding the molecular biology of the biofilms formed by numerous model bacteria. While different bacteria utilize different pathways to build a biofilm, a common feature emerges. An extracellular matrix - generally composed of polysaccharides, proteins, and nucleic acids - holds the biofilm-associated cells together. A full understanding of the processes that control matrix production is required if we are to develop better ways to control and manipulate bacterial biofilms. Over the past two decades our laboratory has focused on studying the process of biofilm formation by the Gram-positive model bacterium Bacillus subtilis. As a result we know possess a deep understanding of the regulatory circuitry that governs matrix production in this organism. The vast majority of our studies have been carried out using small variations on one set of laboratory conditions that were specifically designed to yield very robust biofilms. We are now in a position to take biofilm analyses to the next level. With our current understanding as a starting point, we will now expand our studies to many different environmental conditions. Importantly, we will determine how biofilm formation is influenced by other bacterial species and how it differs when it happen on the surface of a plant's roots. We propose to carry out systems-level analyses of the different responses that B. subtilis mounts during biofilm formation under such diverse condition. Such an approach has not been taken to study biofilms before. To address this gap in knowledge, we will focus our work along the follow four specific aims: 1) Development and Application of New Methodologies to Study B. subtilis Biofilms. 2) A Systems-Level Approach to Understanding Changes in Biofilm Physiology. 3) Interspecies Modulation of Biofilm Formation. 4) The Role of the Environment in Root Colonization and Biofilm Formation.